The present invention relates to the obtaining of bird ES cells, especially to a method of culture and to a medium permitting the culture of these cells.
In effect, in the context of perfecting recombinant protein production technique, the development of a technique of transgenesis [sic] in domestic birds will have extremely important economic repercussions in two major applications:
1. the development of avian strains possessing particular genetic characters (resistance to certain diseases, growth performance features, and the like)
2. the development of recombinant protein production systems in egg albumin.
The biotechnology industry is showing increasing interest in the possibility of producing proteins of interest in biological fluids or organisms (blood, milk, plants, etc.). The production of such proteins in domestic birds"" eggs will certainly constitute a major technological advance in this approach, for several reasons:
numerous mammalian proteins cannot be produced in a mammalian system because their overabundance in these organisms has deleterious effects (for example erythropoietin, which induces pathological hyperglobulinemia in rabbits). Many of these proteins of interest do not display cross-activity with those of birds, thus permitting their overproduction in an avian organism without major pathological effect;
it is very probable that the marketing of recombinant proteins produced in mammals will come up against health problems associated with the presence in this [sic] species of latent organisms which are potentially pathogenic for man (lentiviruses, prions, etc.). This risk is very minimal, not to say almost nonexistent, in relation to pathogenic agents of domestic birds;
the egg constitutes a xe2x80x9ctissuexe2x80x9d which is very dense with respect to a small number of proteins. For example, the major protein of birds"" eggs, ovalbumin, represents 54% of the egg white proteins, equivalent to an average dry weight per egg of 2 grams of dry matter approximately. It is possible reasonably to conceive of producing per egg at least 10% of this mass as recombinant protein. The economic viability is seen to be very great if it is considered that a hen lays on average 2 eggs every three days, and this viability is seen to be much greater than that of large mammals if the much lower breeding costs of domestic birds are considered.
The production of transgenic birds is currently possible at an extremely high cost on account of its very low efficiency. In effect, in birds, the technique of microinjection of DNA into the egg is almost impossible. On the other hand, the use of the vector retrovirus system, the only efficient system to date, remains complex and will certainly come up against a reticence on the part of industrialists on health grounds.
A very great advance in the production of transgenic animals has been brought about in mice by the development of ES cell technology.
ES cells (embryonic stem cells) are totipotent embryonic cells capable of regenerating all the tissues of the embryo, including the germ tissue, after their injection into very early embryos. These cells may hence be considered to be Trojan horses for introducing new genetic information into an animal""s genetic constitution. The possibility of culturing these cells in the long term in vitro affords the possibility of exercising numerous controls before their implantation in vivo. Moreover, these cells may be stored without limit in liquid nitrogen, which constitutes a potential for storage of a genetic constitution.
The use of ES cells nowadays constitutes the most promising approach in domestic birds for the efficient production of transgenic animals.
Recent work from a Canadian group (R. Etches at the Guelph station) has suggested that ES cells must exist in the bird embryo (Petitte et al., 1990). This group at [sic] succeeded in transplanting such cells into embryos and consequently producing animals whose genetic constitution is derived from that of the grafted cells. However, to date, it has not been possible for success to be achieved in culturing these cells in vitro; as a result, it has not been possible to use these cells to transfer a transgene in a stable manner. This is a major impediment to the exploitation of ES cell technology in birds. ES cells may be characterized by three essential types of criteria:
morphology
endogenous alkaline phosphatase activity
reaction with antibodies which are specific for a state of totipotency (ECMA-7, SSEA-1 and SSEA-3, in particular).
To date, it has not been possible to obtain any culture of ES cells which are identified by these collective characteristics.
Accordingly, the subject of the present invention is a culture medium for avian totipotent embryonic cells, of the type containing an avian cell culture medium, characterized in that it contains components supplementary to said avian cell culture medium, said supplementary components being chosen from the group comprising: cytokines, fibroblast growth factors, insulin-like growth factors and stem cell growth factors, and in that it is substantially free from active retinoic acid.
Advantageously, the retinoic acid is substantially inactivated by anti-retinoic acid antibodies (ARMA) present in the medium.
In effect, the media employed often contain serum, in which the amount of endogenous retinoic acid cannot be controlled. On testing the effect on cell differentiation of incorporating in the culture medium an anti-retinoic acid monoclonal antibody which would neutralize the action of retinoic acid, the Applicant found that the presence of this antibody increases the presence in the cultures of cells and colonies having alkaline phosphatase activity.
The cytokine may be chosen, in particular, from LIF, IL-11, IL-6, CNTF and oncostatin M (OSM); advantageously, the cytokines present in the culture medium described above comprise at least one cytokine chosen from the group consisting of LIF, IL-11, IL-6 and the various mixtures thereof, which give the best results for growth stimulation.
Preferably, the fibroblast growth factor is b-FGF (or basic fibroblast growth factor) and the insulin-like growth factor is IFG-1.
The stem cell growth factor (or SCF) is preferably a-SCF (or avian stem cell factor) and m-SCF (or murine stem cell factor).
One of the preferred aspects of the invention relates to a culture medium which contains, besides the basic nutrient components necessary for cell growth, a combination of b-FGF, SCF and LIF. In addition, the presence in the medium of a monoclonal antibody which neutralizes the differentiation activity exerted by retinoic acid increases the number of totipotent embryogenic stem cells.
The presence of a lawn of feeder cells promotes the growth of avian ES cells. Various cell types known to a person skilled in the art may be used; there may be mentioned especially cells such as STO cells, treated with mitomycin or irradiated, BRL-3A cells, LMH cells, QT6 cells and modified QT6 cells such as QT6 Isolde cells, differentiated cells established as a line from cultures of embryonic stem cells induced to differentiate.
STO cells are mouse embryo fibroblasts (ATCC catalogue); BRL-3A cells (ATCC catalogue) are liver cells from xe2x80x9cBuffalo rat liverxe2x80x9d. QT6 cells (ATCC catalogue) and modified QT6 cells such as QT6 Isolde cells are quail fibroblasts (Cosset et al., 1990, J. Virol. 64, 10170-1078) and LMH cells originate from chicken liver carcinoma (Kawagucchi et al., 1987, Cancer Res., 47, 4460-4464).
The culture medium contains, in addition, various essential nutrient components and antibiotics.
A culture medium which is especially suitable for the present invention possesses the following composition:
Advantageously, the bFGF concentration is greater than 5 mg/ml and the IGF-1 concentration is greater than 10 ng/ml.
with the nucleoside stock consisting of the mixture:
and Cos SN representing a culture supernatant of COS-7 cells transfected for transient expression with a vector for the expression of the cDNA of the factor in question, and is suitable for the culture of bird totipotent embryonic cells.
BHK21 medium (or MEM medium) is a culture medium which has been described, in particular, by McPherson, I., and Stoker (1962, Virology 16, 147).
Hepes is hydroxyethylpiperazineethanesulfonate.
According to another aspect, the subject of the invention is a method of culture of avian totipotent embryonic cells (or avian ES cells), characterized in that:
a) cells originating from blastoderm disks of fertilized eggs are suspended in an avian cell culture medium comprising, in addition, at least one compound chosen from cytokines, fibroblast growth factors, insulin-like growth factors and stem cell growth factors, and in which the retinoic acid is substantially inactivated,
b) a lawn of feeder cells or a gelatin-treated culture dish is inoculated with the suspension obtained after step a),
c) the cells are incubated for a specified period,
d) the cells in culture are removed and purified so as to recover bird ES cells.
Preferably, between steps c) and d), one or more additions of fresh medium identical to the one used in step a) are performed at intervals of time.
In one of its embodiments, during step c), a reinoculation of the medium with a cell suspension identical to the suspension prepared in step a) is performed.
The medium of step a) preferably contains the following components: b-FGF, a-SCF, IGF-1, LIF, IL-11, IL-6 and anti-retinoic acid antibodies. According to one of the aspects of the invention, it contains, in addition, the following compounds:
Fetal bovine serum
Chicken serum
Conalbumin
Nonessential amino acids
Sodium pyruvate
Nucleoside stock
Hepes (1M)
xcex2-Mercaptoethanol
Penicillin
Streptomycin
Gentamicin
with the nucleoside stock consisting of the mixture: adenosine, guanosine, cytidine, uridine and thymidine in aqueous solution.
Optionally, during the method of the invention, between steps c) and d), the addition of fresh medium is performed on day 3 and the medium is then changed every day until the next subculturing.
Step d) may be performed, in particular, by enzymatic treatment, washing in a medium not containing any growth factor and centrifugation.
It is possible to collect directly the primary cell cultures, which will then be frozen, or alternatively to produce successive secondary cultures from the cells of the primary culture. In this case, after step d), a step e) is performed in which the ES cells are reinoculated onto a lawn of feeder cells, or onto gelatin-treated dishes, so as to obtain a secondary culture.
Steps d) and e) may be repeated several times in order to have tertiary and successive cultures.
The lawn of feeder cells can consist of various cell types described above, and in particular of mitomycin-treated or irradiated STO cells.
Another of the subjects of the invention is a culture of bird ES cells, or avian ES cells, which are capable of being obtained by the method defined above. A modified avian totipotent embryonic cell can be obtained by integration of the gene coding for a heterologous protein in the genome of an avian ES cell in culture.
Lastly, a method of production of a recombinant protein, characterized in that the gene coding for said protein is integrated in the genome of an avian totipotent embryonic cell in culture, is also included in the invention.
The Applicant has perfected a culture medium and in vitro culture conditions which make it possible to maintain in culture bird cells which possess morphological, kinetic and histochemical properties recalling those of totipotent embryonic cells. These observations have been made with cells derived from both quail and chicken blastoderm disks. The growth of these cells in culture in vitro is made possible by the perfecting of a novel medium especially adapted to the culture of bird embryonic cells. The presence, maintenance and propagation of totipotent cells in culture are known to permit their injection into recipient embryos. The contribution to the morphogenesis of the somatic and germ tissues in the recipient animals as a result of a totipotent character may lead to the obtaining of transgenic animals.